Dual-Capped Helical Interface Mimics.

Disruption of protein-protein interactions is medicinally important. Interface helices may be mimicked in helical probes featuring enhanced rigidities, binding to protein targets, stabilities in serum, and cell uptake. This form of mimicry is dominated by stapling between side chains of helical residues: there has been less progress on helical N-caps, and there were no generalizable C-caps. Conversely, in natural proteins, helicities are stabilized and terminated by C- and N-caps but not staples. Bicyclic caps previously introduced by us enable interface helical mimicry featuring rigid synthetic caps at both termini in this work. An unambiguously helical dual-capped system proved to be conformationally stable, binding cyclins A and E, and showed impressive cellular uptake. In addition, the dual-capped mimic was completely resistant to proteolysis in serum over an extended period when compared with "gold standard" hydrocarbon-stapled controls. Dual-capped peptidomimetics are a new, generalizable paradigm for helical interface probe design.

Testing the ectomycorrhizal-dominance hypothesis for ecosystem multifunctionality in a subtropical mountain forest.

Mycorrhizal associations are key mutualisms that shape the structure of forest communities and multiple ecosystem functions. However, we lack a framework for predicting the varying dominance of distinct mycorrhizal associations in an integrated proxy of multifunctionality across ecosystems. Here, we used the datasets containing diversity of mycorrhizal associations and 18 ecosystem processes related to supporting, provisioning, and regulating services to examine how the dominance of ectomycorrhiza (EcM) associations affects ecosystem multifunctionality in subtropical mountain forests in Southwest China. Meanwhile, we synthesized the prevalence of EcM-dominant effects on ecosystem functioning in forest biomes. Our results demonstrated that elevation significantly modified the distributions of EcM trees and fungal dominance, which in turn influenced multiple functions simultaneously. Multifunctionality increased with increasing proportion of EcM associations, supporting the ectomycorrhizal-dominance hypothesis. Meanwhile, we observed that the impacts of EcM dominance on individual ecosystem functions exhibited different relationships among forest biomes. Our findings highlight the importance of ectomycorrhizal dominance in regulating multifunctionality in subtropical forests. However, this ectomycorrhizal feedback in shaping ecosystem functions cannot necessarily be generalized across forests. Therefore, we argue that the predictions for ecosystem multifunctionality in response to the shifts of mycorrhizal composition could vary across space and time.

A fluorescent electrophile for CLIPS: self indicating TrkB binders.

Combination of cysteine-containing peptides with electrophiles provides efficient access to cyclo-organopeptides. However, there are no routes to intrinsically fluorescent cyclo-organopeptides containing robust, brilliant fluorophores emitting at wavelengths longer than cellular autofluorescence. We show such fluorescent cyclo-organopeptides can be made via SNAr reactions of cysteine-containing peptides with a BODIPY system. Seven compounds of this type were prepared to test as probes; six contained peptide sequences corresponding to loop regions in brain-derived neurotrophic factor and neurotrophic factor 4 (BDNF and NT-4) which bind tropomyocin receptor kinase B (TrkB). Cellular assays in serum-free media indicated two of the six key compounds induced survival of HEK293 cells stably transfected with TrkB whereas a control did not. The two compounds inducing cell survival bound TrkB on those cells (Kd ∼40 and 47 nM), illustrating how intrinsically fluorescent cyclo-organopeptides can be assayed for quantifiable binding to surface receptors in cell membrane environments.

PROTAC-mediated CDK degradation differentially impacts cancer cell cycles due to heterogeneity in kinase dependencies.

BACKGROUND: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibition yields differential cellular responses in multiple tumor models due to redundancy in cell cycle. We investigate whether the differential requirements of CDKs in multiple cell lines function as determinant of response to pharmacological agents that target these kinases. METHODS: We utilized proteolysis-targeted chimeras (PROTACs) that are conjugated with palbociclib (Palbo-PROTAC) to degrade both CDK4 and CDK6. FN-POM was synthesized by chemically conjugating pomalidomide moiety with a multi-kinase inhibitor, FN-1501. Patient derived PDAC organoids and PDX model were utilized to investigate the effect of FN-POM in combination with palbociclib. RESULTS: Palbo-PROTAC mediates differential impact on cell cycle in different tumor models, indicating that the dependencies to CDK4 and 6 kinases are heterogenous. Cyclin E overexpression uncouples cell cycle from CDK4/6 and drives resistance to palbo-PROTAC. Elevated expression of P16INK4A antagonizes PROTAC-mediated degradation of CDK4 and 6. FN-POM degrades cyclin E and CDK2 and inhibits cell cycle progression in P16INK4A-high tumor models. Combination of palbociclib and FN-POM cooperatively inhibit tumor cell proliferation via RB activation. CONCLUSION: Resistance to CDK4/6 inhibition could be overcome by pharmacologically limiting Cyclin E/CDK2 complex and proves to be a potential therapeutic approach.

Community-wide patterns in pollen and ovule production, their ratio (P/O), and other floral traits along an elevation gradient in southwestern China.

BACKGROUND: As the male and female gametophytes of flowering plants, pollen and ovules largely determine the upper and lower boundaries of plant reproductive success. It is commonly predicted that pollen and ovule number per flower should increase, and pollen-ovule ratio (P/O) per flower should decrease with increasing elevation in response to a more stochastic pollination environment. Here, we aimed to determine the response of pollen number, ovule number, and P/O to other floral traits and elevation gradients for 84 insect-pollinated herbaceous flowering plant species in five sub-alpine and alpine communities (2709 to 3896 m a.s.l.) on Yulong Snow Mountain, southwestern China. RESULTS: Six floral traits, including P/O, floral display area, flower number, tube depth, flower shape, and pollen presentation, were highly correlated with pollen and ovule number per flower. With increasing elevation, pollen number and P/O per flower increased marginally and significantly, respectively; ovule number per individual, flower number per individual, stigma stamen separation, and inflorescence height decreased significantly. However, ovule number per flower and other floral traits (i.e., floral display area, tube depth, stigma height, stamen height, and pollen and P/O per individual) did not change with elevation. We detected significant phylogenetic signals for pollen number, ovule number, and P/O, suggesting that these traits may be highly conserved and with limited response to changing environmental conditions. CONCLUSIONS: Results revealed patterns of plant reproductive character evolution along elevation gradients and the potential factors governing their spatial variation in high-elevation environments. Plant species at high elevations are more likely adapted to cross-pollination, indicated by increased P/O per flower at high elevations on Yulong Mountain. Combined effects of phylogenetic history and plant-pollinator interactions should determine plant trait evolution.

Plastome phylogenomics and morphological traits analyses provide new insights into the phylogenetic position, species delimitation and speciation of Triplostegia (Caprifoliaceae).

BACKGROUND: The genus Triplostegia contains two recognized species, T. glandulifera and T. grandiflora, but its phylogenetic position and species delimitation remain controversial. In this study, we assembled plastid genomes and nuclear ribosomal DNA (nrDNA) cistrons sampled from 22 wild Triplostegia individuals, each from a separate population, and examined these with 11 recently published Triplostegia plastomes. Morphological traits were measured from herbarium specimens and wild material, and ecological niche models were constructed. RESULTS: Triplostegia is a monophyletic genus within the subfamily Dipsacoideae comprising three monophyletic species, T. glandulifera, T. grandiflora, and an unrecognized species Triplostegia sp. A, which occupies much higher altitude than the other two. The new species had previously been misidentified as T. glandulifera, but differs in taproot, leaf, and other characters. Triplotegia is an old genus, with stem age 39.96 Ma, and within it T. glandulifera diverged 7.94 Ma. Triplostegia grandiflora and sp. A diverged 1.05 Ma, perhaps in response to Quaternary climate fluctuations. Niche overlap between Triplostegia species was positively correlated with their phylogenetic relatedness. CONCLUSIONS: Our results provide new insights into the species delimitation of Triplostegia, and indicate that a taxonomic revision of Triplostegia is needed. We also identified that either rpoB-trnC or ycf1 could serve as a DNA barcode for Triplostegia.

Plants, pollinators and their interactions under global ecological change: The role of pollen DNA metabarcoding.

Anthropogenic activities are triggering global changes in the environment, causing entire communities of plants, pollinators and their interactions to restructure, and ultimately leading to species declines. To understand the mechanisms behind community shifts and declines, as well as monitoring and managing impacts, a global effort must be made to characterize plant-pollinator communities in detail, across different habitat types, latitudes, elevations, and levels and types of disturbances. Generating data of this scale will only be feasible with rapid, high-throughput methods. Pollen DNA metabarcoding provides advantages in throughput, efficiency and taxonomic resolution over traditional methods, such as microscopic pollen identification and visual observation of plant-pollinator interactions. This makes it ideal for understanding complex ecological networks and their responses to change. Pollen DNA metabarcoding is currently being applied to assess plant-pollinator interactions, survey ecosystem change and model the spatiotemporal distribution of allergenic pollen. Where samples are available from past collections, pollen DNA metabarcoding has been used to compare contemporary and past ecosystems. New avenues of research are possible with the expansion of pollen DNA metabarcoding to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for understanding the evolutionary and ecological processes that support biodiversity, and predicting and responding to the impacts of change.

Cyanine Dye Conjugation Enhances Crizotinib Localization to Intracranial Tumors, Attenuating NF-κB-Inducing Kinase Activity and Glioma Progression.

Glioblastoma (GBM) is a highly aggressive form of brain cancer with a poor prognosis and limited treatment options. The ALK and c-MET inhibitor Crizotinib has demonstrated preclinical therapeutic potential for newly diagnosed GBM, although its efficacy is limited by poor penetration of the blood brain barrier. Here, we identify Crizotinib as a novel inhibitor of nuclear factor-κB (NF-κB)-inducing kinase, which is a key regulator of GBM growth and proliferation. We further show that the conjugation of Crizotinib to a heptamethine cyanine dye, or a near-infrared dye (IR-Crizotinib), attenuated glioma cell proliferation and survival in vitro to a greater extent than unconjugated Crizotinib. Moreover, we observed increased IR-Crizotinib localization to orthotopic mouse xenograft GBM tumors, which resulted in impaired tumor growth in vivo. Overall, IR-Crizotinib exhibited improved intracranial chemotherapeutic delivery and tumor localization with concurrent inhibition of NIK and noncanonical NF-κB signaling, thereby reducing glioma growth in vitro, as well as in vivo, and increasing survival in a preclinical rodent model.

Synthetic helical peptide capping strategies.

Relatively small mimics of interface secondary structures can be used to disrupt protein-protein interactions (PPIs). This strategy is valuable because many PPIs are pivotal in cell biology and contemporary medicinal chemistry. Small peptides tend to have random coil conformations in solution, so the entropy costs are high for them to order into states binding protein receptors. Consequently, peptides constrained in conformations resembling interface secondary structures are favored for enhanced affinities to PPI components. Helices are commonly found at PPI interfaces. The two general strategies that have emerged for imposing helical constraints in probes, capping and stapling, are often confused because both involve formation of macrocyclic rings. This review considers parameters that distinguish capping from stapling. Capping motifs terminate helices and project the adjacent peptide units in non-helical orientations, but stapling enforces helical motifs in ways that enable adjacent peptide fragments to extend helices. There is no evidence that stapling is more effective than capping for helix mimicry, but stapling is used more frequently. This imbalance may be because no strategies have emerged for synthetic C-capping with compact unit; if convenient and effective C-capping strategies were available then capping strategies should be more widely used.

Streamlined Protein-Protein Interface Loop Mimicry.

Cyclic peptides comprising endocyclic organic fragments, "cyclo-organopeptides", can be probes for perturbing protein-protein interactions (PPIs). Finding loop mimics is difficult because of high conformational variability amongst targets. Backbone Matching (BM), introduced here, helps solve this problem in the illustrative cases by facilitating efficient evaluation of virtual cyclo-organopeptide core-structure libraries. Thus, 86 rigid organic fragments were selected to build a library of 602 cyclo-organopeptides comprising Ala and organic parts: "cyclo-{-(Ala)n -organo-}". The central hypothesis is "hit" library members have accessible low energy conformers corresponding to backbone structures of target protein loops, while library members which cannot attain this conformation are probably unworthy of further evaluation. BM thereby prioritizes candidate loop mimics, so that less than 10 cyclo-organopeptides are needed to be prepared to find leads for two illustrative PPIs: iNOS ⋅ SPSB2, and uPA ⋅ uPAR.

Antibody-dependent cellular phagocytosis of tropomyosin receptor kinase C (TrkC) expressing cancer cells for targeted immunotherapy.

Conventional cancer therapies such as chemotherapy are non-selective and induce immune system anergy, which lead to serious side effects and tumor relapse. It is a challenge to prime the body's immune system in the cancer-bearing subject to produce cancer antigen-targeting antibodies, as most tumor-associated antigens are expressed abundantly in cancer cells and some of normal cells. This study illustrates how hapten-based pre-immunization (for anti-hapten antibodies production) combined with cancer receptor labeling with hapten antigen constructs can elicit antibody-dependent cellular phagocytosis (ADCP). Thus, the hapten antigen 2,4-dinitrophenol (DNP) was covalently combined with a cancer receptor-binding dipeptide (IYIY) to form a dipeptide-hapten construct (IYIY-DNP, MW = 1322.33) that targets the tropomyosin receptor kinase C (TrkC)-expressed on the surface of metastatic cancer cells. IYIY-DNP facilitated selective association of RAW264.7 macrophages to the TrkC expressing 4T1 cancer cells in vitro, forming cell aggregates in the presence of anti-DNP antibodies, suggesting initiation of anti-DNP antibody-dependent cancer cell recognition of macrophages by the IYIY-DNP. In in vivo, IYIY-DNP at 10 mg/kg suppressed growth of 4T1 tumors in DNP-immunized BALB/c mice by 45% (p < 0.05), when comparing the area under the tumor growth curve to that of the saline-treated DNP-immunized mice. Meanwhile, IYIY-DNP at 10 mg/kg had no effect on TrkC-negative 67NR tumor-bearing mice immunized with DNP. Tumor growth suppression activity of IYIY-DNP in DNP-immunized mice was associated with an increase in the anti-DNP IgG (7.3 × 106 ± 1.6 U/mL) and IgM (0.9 × 106 ± 0.07 U/mL) antibodies after five cycles of DNP treatment, demonstrated potential for hapten-based pre-immunization then treatment with IYIY-DNP to elicit ADCP for improved immunotherapy of TrkC expressing cancers.

Hows and Whys of Tumor-Seeking Dyes.

Active targeting uses molecular fragments that bind receptors overexpressed on cell surfaces to deliver cargoes, and this selective delivery to diseased over healthy tissue is valuable in diagnostic imaging and therapy. For instance, targeted near-infrared (near-IR) dyes can mark tissue to be excised in surgery, and radiologists can use active targeting to concentrate agents for positron emission tomography (PET) in tumor tissue to monitor tumor metastases. Selective delivery to diseased tissue is also valuable in some treatments wherein therapeutic indexes (toxic/effective doses) are key determinants of efficacy. However, active targeting will only work for cells expressing the pivotal cell surface receptor that is targeted. That is a problem because tumors, even ones derived from the same organ, are not homogeneous, patient-to-patient variability is common, and heterogeneity can occur even in the same patient, so monotherapy with one actively targeted agent is unlikely to be uniformly effective. A particular category of fluorescent heptamethine cyanine-7 (Cy-7) dyes, here called tumor seeking dyes, offer a way to circumvent this problem because they selectively accumulate in any solid tumor. Furthermore, they persist in tumor tissue for several days, sometimes longer than 72 h. Consequently, tumor seeking dyes are near-IR fluorescent targeting agents that, unlike mAbs (monoclonal antibodies), accumulate in any solid lesion, thus overcoming tumor heterogeneity, and persist there for long periods, circumventing the rapid clearance problems that bedevil low molecular mass drugs. Small molecule imaging agents and drugs attached to tumor-seeking dyes have high therapeutic indices and long residence times in cancer cells and tumor tissue. All this sounds too good to be true. We believe most of this is true, but the controversy is associated with how and why these characteristics arise. Prior to our studies, the prevailing hypothesis, often repeated, was that tumor seeking dyes are uptaken by organic anion transporting polypeptides (OATPs) overexpressed on cancer cells. This Account summarizes evidence indicating tumor seeking Cy-7 dyes have exceptional accumulation and persistence properties because they covalently bind to albumin in vivo. That adduct formation provides a convenient way to form albumin-bound pharmaceuticals labeled with near-IR fluorophores which can be tracked in vivo. This understanding may facilitate more rapid developments of generally applicable actively targeted reagents.

Small molecule peptidomimetic trypsin inhibitors: validation of an EKO binding mode, but with a twist.

Examination of a series of naturally-occurring trypsin inhibitor proteins, led to identification of a set of three residues (which we call the "interface triplet") to be determinant of trypsin binding affinity, hence excellent templates for small molecule mimicry. Consequently, we attempted to use the Exploring Key Orientation (EKO) strategy developed in our lab to evaluate small molecules that mimic the interface triplet regions of natural trypsin inhibitors, and hence potentially might bind and inhibit the catalytic activity of trypsin. A bis-triazole scaffold ("TT-mer") was the most promising of the molecules evaluated in silico. Twelve such compounds were synthesized and assayed against trypsin, among which the best showed a Kd of 2.1 µM. X-ray crystallography revealed a high degree of matching between an illustrative TT-mer's actual binding mode and that of the mimics that overlaid the interface triplet in the crystal structure. Deviation of the third side chain from the PPI structure seems to be due to alleviation of an unfavorable dipole-dipole interaction in the small molecule's actual bound conformation.

Fluorescent kinase inhibitors as probes in cancer.

Fluorescent dyes attached to kinase inhibitors (KIs) can be used to probe kinases in vitro, in cells, and in vivo. Ideal characteristics of the dyes vary with their intended applications. Fluorophores used in vitro may inform on kinase active site environments, hence the dyes used should be small and have minimal impact on modes of binding. These probes may have short wavelength emissions since blue fluorophores are perfectly adequate in this context. Thus, for instance, KI fragments that mimic nucleobases may be modified to be fluorescent with minimal perturbation to the kinase inhibitor structure. However, progressively larger dyes, that emit at longer wavelengths, are required for cellular and in vivo work. In cells, it is necessary to have emissions above autofluorescence of biomolecules, and near infrared dyes are needed to enable excitation and observation through tissue in vivo. This review is organized to describe probes intended for applications in vitro, in cells, then in vivo. The readers will observe that the probes featured tend to become larger and responsive to the near infared end of the spectrum as the review progresses. Readers may also be surprised to realize that relatively few dyes have been used for fluorophore-kinase inhibitor conjugates, and the area is open for innovations in the types of fluorophores used.

A, B, C's of Trk Receptors and Their Ligands in Ocular Repair.

Neurotrophins are a family of closely related secreted proteins that promote differentiation, development, and survival of neurons, which include nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. All neurotrophins signal through tropomyosin receptor kinases (TrkA, TrkB, and TrkC) which are more selective to NGF, brain-derived neurotrophic factor, and neurotrophin-3, respectively. NGF is the most studied neurotrophin in the ocular surface and a human recombinant NGF has reached clinics, having been approved to treat neurotrophic keratitis. Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 are less studied neurotrophins in the ocular surface, even though brain-derived neurotrophic factor is well characterized in glaucoma, retina, and neuroscience. Recently, neurotrophin analogs with panTrk activity and TrkC selectivity have shown promise as novel drugs for treating dry eye disease. In this review, we discuss the biology of the neurotrophin family, its role in corneal homeostasis, and its use in treating ocular surface diseases. There is an unmet need to investigate parenteral neurotrophins and its analogs that activate TrkB and TrkC selectively.

Natural hybridization among three Rhododendron species (Ericaceae) revealed by morphological and genomic evidence.

BACKGROUND: Natural hybridization can influence the adaptive response to selection and accelerate species diversification. Understanding the composition and structure of hybrid zones may elucidate patterns of hybridization processes that are important to the formation and maintenance of species, especially for taxa that have experienced rapidly adaptive radiation. Here, we used morphological traits, ddRAD-seq and plastid DNA sequence data to investigate the structure of a Rhododendron hybrid zone and uncover the hybridization patterns among three sympatric and closely related species. RESULTS: Our results show that the hybrid zone is complex, where bi-directional hybridization takes place among the three sympatric parental species: R. spinuliferum, R. scabrifolium, and R. spiciferum. Hybrids between R. spinuliferum and R. spiciferum (R. ×duclouxii) comprise multiple hybrid classes and a high proportion of F1 generation hybrids, while a novel hybrid taxon between R. spinuliferum and R. scabrifolium dominated the F2 generation, but no backcross individuals were detected. The hybrid zone showed basically coincident patterns of population structure between genomic and morphological data. CONCLUSIONS: Natural hybridization exists among the three Rhododendron species in the hybrid zone, although patterns of hybrid formation vary between hybrid taxa, which may result in different evolutionary outcomes. This study represents a unique opportunity to dissect the ecological and evolutionary mechanisms associated with adaptive radiation of Rhododendron species in a biodiversity hotspot.

Piptides: New, Easily Accessible Chemotypes For Interactions With Biomolecules.

Small molecule probe development is pivotal in biomolecular science. Research described here was undertaken to develop a non-peptidic chemotype, piptides, that is amenable to convenient, iterative solid-phase syntheses, and useful in biomolecular probe discovery. Piptides can be made from readily accessible pip acid building blocks and have good proteolytic and pH stabilities. An illustrative application of piptides against a protein-protein interaction (PPI) target was explored. The Exploring Key Orientations (EKO) strategy was used to evaluate piptide candidates for this. A library of only 14 piptides contained five members that disrupted epidermal growth factor (EGF) and its receptor, EGFR, at low micromolar concentrations. These piptides also caused apoptotic cell death, and antagonized EGF-induced phosphorylation of intracellular tyrosine residues in EGFR.

Complete plastome sequencing resolves taxonomic relationships among species of Calligonum L. (Polygonaceae) in China.

BACKGROUND: Calligonum (Polygonaceae) is distributed from southern Europe through northern Africa to central Asia, and is typically found in arid, desert regions. Previous studies have revealed that standard DNA barcodes fail to discriminate Calligonum species. In this study, the complete plastid genomes (plastome) for 32 accessions of 21 Calligonum species is sequenced to not only generate the first complete plastome sequence for the genus Calligonum but to also 1) Assess the ability of the complete plastome sequence to discern species within the group, and 2) screen the plastome sequence for a cost-effective DNA barcode that can be used in future studies to resolve taxonomic relationships within the group. RESULTS: The whole plastomes of Calligonum species possess a typical quadripartite structure. The size of the Calligonum plastome is approximately 161 kilobase pairs (kbp), and encodes 113 genes, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Based on ML phylogenetic tree analyses, the complete plastome has higher species identification (78%) than combinations of standard DNA barcodes (rbcL + matK + nrITS, 56%). Five newly screened gene regions (ndhF, trnS-G, trnC-petN, ndhF-rpl32, rpl32-trnL) had high species resolution, where ndhF and trnS-G were able to distinguish the highest proportion of Calligonum species (56%). CONCLUSIONS: The entire plastid genome was the most effective barcode for the genus Calligonum, although other gene regions showed great potential as taxon-specific barcodes for species identification in Calligonum.

Role of Albumin in Accumulation and Persistence of Tumor-Seeking Cyanine Dyes.

Some heptamethine cyanine dyes accumulate in solid tumors in vivo and persist there for several days. The reasons why they accumulate and persist in tumors were incompletely defined, but explanations based on uptake into cancer cells via organic anion transporting polypeptides (OATPs) have been widely discussed. All cyanine-based "tumor-seeking dyes" have a chloride centrally placed on the heptamethine bridge (a "meso-chloride"). We were intrigued and perplexed by the correlation between this particular functional group and tumor uptake, so the following study was designed. It features four dyes (1-Cl, 1-Ph, 5-Cl, and 5-Ph) with complementary properties. Dye 1-Cl is otherwise known as MHI-148, and 1-Ph is a close analog wherein the meso-chloride has been replaced by a phenyl group. Data presented here shows that both 1-Cl and 1-Ph form noncovalent adducts with albumin, but only 1-Cl can form a covalent one. Both dyes 5-Cl and 5-Ph have a methylene (CH2) unit replaced by a dimethylammonium functionality (N+Me2). Data presented here shows that both these dyes 5 do not form tight noncovalent adducts with albumin, and only 5-Cl can form a covalent one (though much more slowly than 1-Cl). In tissue culture experiments, uptake of dyes 1 is more impacted by the albumin in the media than by the pan-OATP uptake inhibitor (BSP) that has been used to connect uptake of tumor-seeking dyes in vivo with the OATPs. Uptake of 1-Cl in media containing fluorescein-labeled albumin gave a high degree of colocalization of intracellular fluorescence. No evidence was found for the involvement of OATPs in uptake of the dyes into cells in media containing albumin. In an in vivo tumor model, only the two dyes that can form albumin adducts (1-Cl and 5-Cl) gave intratumor fluorescence that persisted long enough to be clearly discerned over the background (∼4 h); this fluorescence was still observed at 48 h. Tumors could be imaged with a higher contrast if 5-Cl is used instead of 1-Cl, because 5-Cl is cleared more rapidly from healthy tissues. Overall, the evidence is consistent with in vitro and in vivo results and indicates that the two dyes in the test series that accumulate in tumors and persist there (1-Cl and 5-Cl, true tumor-seeking dyes) do so as covalent albumin adducts trapped in tumor tissue via uptake by some cancer cells and via the enhanced permeability and retention (EPR) effect.

Unconventional Secondary Structure Mimics: Ladder-Rungs.

Secondary structures tend to be recognizable because they have repeating structural motifs, but mimicry of these does not have to follow such well-defined patterns. Bioinformatics studies to match side-chain orientations of a novel hydantoin triazole chemotype (1) to protein-protein interfaces revealed it tends to align well across parallel and antiparallel sheets, like rungs on a ladder. One set of these overlays was observed for the protein-protein interaction uPA⋅uPAR. Consequently, chemotype 1 was made with appropriate side-chains to mimic uPA at this interface. Biophysical assays indicate these compounds did in fact bind uPAR, and elicit cellular responses that affected invasion, migration, and wound healing.